[Analysis of ethambutol drug-resistance gene mutation in Mycobacterium tuberculosis by specific endonuclease].

OBJECTIVE: Surveyor nuclease is a member of the family of plant endonucleases that cleave heteroduplexes DNA with high specificity at sites of base substitution mismatch and DNA distortion. Heteroduplex analysis by Surveyor nuclease is a relatively new method. The purpose of this study was to explore the application of this method in analysis of drug-resistance gene mutation in Mycobacterium tuberculosis. METHODS: Surveyor nuclease was used to analyze embB gene mutation of 60 Mycobacterium tuberculosis isolates, among which 45 were EMB-resistant and embB gene mutation was found by sequencing, and 15 EMB-susceptible isolates without embB mutation. PCR amplification was carried out first, and then hybridized with products of H37Rv to form heteroduplex, cleaved by Surveyor nuclease, and lastly the results were shown by polyacrylamide gel electrophoresis and mutation was judged according to electrophoresis profile. RESULTS: Forty-five EMB-resistant isolates were found harboring embB gene mutation, while in the 15 EMB-susceptible isolates no mutation was found. All the 45 EMB-resistant isolates were revealed to harbor embB gene hotspot codon 306 mutation, among which 33 with ATG-->GTG, 3 with ATG-->ATT, 5 with ATG-->ATA, 2 with ATG-->ATC, 2 with ATG-->CTG. These results from Surveyor nuclease method was completely parallel to those of sequencing. CONCLUSION: Heteroduplex analysis by Surveyor nuclease may become a useful method for drug-resistance gene mutation analysis for Mycobacterium tuberculosis because of its simplicity, stability, low-cost, and high sensitivity and specificity.

[Impact of anti-tuberculosis drug resistance on treatment outcome of pulmonary tuberculosis patients receiving directly observed treatment strategy in Henan Province, China].

OBJECTIVE: To study the impact of anti-tuberculosis drug resistance on treatment outcome of pulmonary tuberculosis (TB) patients receiving directly observed treatment strategy (DOTS) in Henan Province, China. METHODS: From Aug. 2001 to Jun. 2002, the second round surveillance of anti-TB drug resistance was carried out in cooperation with WHO in Henan Province, China. The proportion method was used for drug susceptibility test and all enrolled patients were administrated with DOTS. RESULTS: The treatment success rate of smear-positive patients was 85.5% (1,343/1,571), of which the treatment success rate of initial-treatment cases was 89.6% (1,159/1,293). The treatment success rates among 565 anti-TB drug resistant cases and 215 multi-drug-resistant-TB (MDR-TB) cases were 76.6% (433/565), (initial treatment cases: 86.8%, 341/393; re-treated cases: 53.5%, 92/172; being significantly different), 58.6% (126/215), (initial treatment cases: 75.4%, 83/110; re-treated cases: 41.0%, 43/105; being significantly different), respectively. For the cases being resistant to 2, 3 or 4 drugs, the treatment success rates in initial treatment cases were all higher than those in re-treatment cases. Multivariate analysis showed that re-treatment, resistance to 2 plus drugs or MDR were the significant risk factors for treatment failure, and that re-treatment, older age and MDR were the significant risk factors for TB death. CONCLUSION: The treatment outcome of initial treatment of smear-positive patients with DOTS in Henan was satisfying, but that of retreated patients, especially retreated patients of MDR was very poor. Re-treatment, older age, resistance to 2 anti-TB drugs or MDR were the risk factors for the poor outcome of treatment with DOTS.

Genotypic analysis of multidrug-resistant Mycobacterium tuberculosis isolates recovered from central China.

DNA sequencing analysis was used to investigate genetic alterations in the rpoB, katG, and inhA regulatory region and embB in 66 Mycobacterium tuberculosis isolates recovered from Central China. Of the 36 multidrug-resistant isolates, 33 (92%) had mutations in the amplified region of rpoB. The most frequent mutation (58%, 19/36) was S531L (TCG-->TTG). At least one mutation was found in the katG and inhA regulatory region in 83% (30/36) of the multidrug-resistant isolates, and mutations at katG codon 315 were identified in 78% (28/36). Alterations at embB306 may not confer resistance to EMB, and embB306 mutants were more frequently accompanied by rpoB mutations (100%, 16/16) than by katG 315 mutations (75%, 12/16). Our results show that geographic variation in the molecular genetic mechanism is responsible for drug resistance in multidrug-resistant M. tuberculosis. This observation will facilitate the development of a rapid molecular drug resistance screening approach for drug-resistant M. tuberculosis.